The concept of using RNA molecules as therapeutic agents has recently received increased attention with the identification of RNA interference (RNAi). RNAi has emerged as a powerful tool to silence gene expression in multiple organisms. Recent research has demonstrated the utility of RNAi in inhibiting the replication of viruses such as HIV-1. Sequence-specific degradation of viral or cellular RNA has been achieved using small, dsRNA gene silencing triggers, termed small interfering RNAs (siRNAs). To introduce RNAi as a therapeutic modality against HIV-1 it must durably control the rapid replication kinetics and high degree of variation of HIV-1. We have previously demonstrated the emergence and selection of viral quasispecies resistant to siRNA targeting the HIV-1 transactivator protein tat. To counteract the rapid emergence of resistance, we hypothesize that co-expression of multiple siRNAs targeting conserved viral RNA sequences as well as mRNA sequences of critical host genes (e.g. CCR5) may reduce the emergence of siRNA resistant virus. The goal of this research proposal is to achieve intracellular antiretroviral immunity by DNA vectors, which express multiple siRNAs against HIV-1 RNA and co-receptor CCR5 delivered by a microRNA cluster. The specific aims of this study are: 1. To engineer pre-micro-siRNAs against HIV-1 and CCR5. 2. To generate AAV-DNA vectors co-expressing multiple pre-micro-siRNAs targeting different viral and host genes. 3. To determine the antiviral activity and durability of HIV-1 inhibition by the expression of multiple siRNAs in stable cell lines and primary lymphocytes.